Many analytical methods require side-by-side or sequential comparison to quantitatively compare differences between two samples. For example, to compare proteins on a Western blot, samples are run on adjacent lanes; to compare small molecules on HPLC, samples are run sequentially one next to the others. This format of analysis requires twice the effort in sample preparation; in addition, variability can be introduced into the system if the treatments of one sample slightly deviated from another. For these reasons, many repetitions plus statistical analysis are required before any differences found become credible. A method that allows two samples to be mixed together for analysis and quantitative comparison will provide significant advantages in analytical precision.
Existing Methods:
Multiplexing methodologies have been around for a while especially the use of different color dyes for comparative labeling. For example, a tissue section can be used for simultaneous multiple immuno-histochemistry experiment by using antibody conjugated to different color dyes. The intensity of each dye is used to compare the relative abundance of each type of antigens that the different color-coded antibodies bind to.
Recently fluorescent dyes of different colors are used to label DNA, RNA, and even proteins to enable two or more of these samples to be mixed together and analyzed simultaneously. One application is a DNA array where DNA or RNA from different samples are labeled with dyes that fluoresce at two different wavelength, mixed, applied to the same array for competitive binding and then read to see which sample has more of which genes expressed. Another important application is the labeling of two proteins samples by different fluorescent colors dyes such as Cydyes sold by Amersham Biosciences and Alexa fluor dyes sold by Molecular Probes. These protein samples are then mixed and co-separated by 2-dimensional gel electrophoresis into thousands of dots based on the proteins' differences in isoelectric points and apparent molecular weights. A fluorescent scanner is used to read Cy2, Cy3 or Cy5 signal separately and computer software compare the signal intensity between them for quantitative comparison of protein abundance between the samples.
Labeling and Detection
The current labeling techniques for fluorescent labeling require coupling of bulky and disruptive fluorochromes to amino acids or nucleotides within the molecule of interest to facilitate detection. While DNA and RNA often can accommodate structural modifications caused by covalent linkage with dyes for DNA array analysis, proteins cannot for protein array analysis. A fluorochrome such as Cydye often will modify amino acids, such as lysine, and will effectively change the epitope structure that the lysine is involved in. As a result, the label interferes with the analyte and often prevents an antibody from binding normally to labeled proteins the same way it would to unlabeled proteins. Furthermore, dye may alter the structure of DNA or RNA significantly, such that proteins, e.g., transcription factors, won't be able to recognize and bind as they would do with native sequences.
Radioactive labeling solves the problem by making the labeling group much smaller, or better yet, by isotope replacement with identical atoms. Additionally, radioactive labeling is at least 100 times more sensitive than many other methods upon detection. Replacement labeling can be done with direct incorporation of labeled precursors such as amino acids yielding labeled proteins with absolutely no chemical modification. Such metabolic labeling techniques are done routinely in research laboratories. Once labeled, X-ray photography can be used to detect and record the respective radiation. Since X-ray film has a limited linear range thus usually not used for quantitative analysis, other methods have evolved over time. A commonly used method comprises the use of a phosphorescent storage imaging screen. The high-energy radiation excites the storage material's electrons into their phosphoresced state at which they will remain until excited again by the right quanta of energy. Using that principle, a phosphorescent screen captures some of the radiation energy, stores it, and gives back when read with a tuned laser. Such devices are also commonly used in photography. Another method that enables quantitative analysis of radiation is Scintillation Counting. A scintillation material is mixed with the radioactive sample so that when the material is struck with high-energy radiation it will give up light for easy detection and quantification. Direct detection with devices such as Geiger counter is also possible; however, the cost for making such instruments has limited its use.